This study investigated the non-thermal effects of Wi-Fi radiofrequency radiation of 2.4 GHz on global gene expression in Escherichia coli K-12 DH5α. High-throughput RNA-sequencing of 2.4 GHz exposed and non-exposed bacteria revealed that 101 genes were differentially expressed (DEGs) at P ≤ 0.05. The up-regulated genes were 52 while the down-regulated ones were 49. QRT-PCR analysis of pgaD, fliC, cheY, malP, malZ, motB, alsC, alsK, appB and appX confirmed the RNA-seq results. About 7% of DEGs are involved in cellular component organization, 6% in response to stress stimulus, 6% in biological regulation, 6% in localization, 5% in locomotion and 3% in cell adhesion. Database for annotation, visualization and integrated discovery (DAVID) functional clustering revealed that DEGs with high enrichment score included genes for localization of cell, locomotion, chemotaxis, response to external stimulus and cell adhesion. Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis showed that the pathways for flagellar assembly, chemotaxis and two-component system were affected. Go enrichment analysis indicated that the up-regulated DEGs are involved in metabolic pathways, transposition, response to stimuli, motility, chemotaxis and cell adhesion. The down-regulated DEGs are associated with metabolic pathways and localization of ions and organic molecules. Therefore, the exposure of E. coli DH5α to Wi-Fi radiofrequency radiation for 5 hours influenced several bacterial cellular and metabolic processes.